Molecular mechanisms utilized by axons to ensure proper wiring in the brain involve multiple ligand-receptor interactions, as extracellular guidance cues, such as netrins and RGMs (repulsive guidance molecule), are critical in the process of axon path-finding. Receptors for netrins involved in axon path-finding include DCC (deleted in colorectal cancer) and neogenin, a family of immunoglobin domain-containing proteins. Neogenin is also a receptor for RGMs. The long-term goal of our work is to elucidate intracellular signaling mechanisms initiated by DCC and neogenin engagement. In this regard, the PI's laboratory has identified binding partners for DCC and neogenin which are likely to participate in netrin-1/RGM-induced intracellular signaling, including PITPalpha (phosphatidylinositol transfer protein alpha), a protein essential for ),), ( phosphatidylinositol signaling, and myosin X (Myo X), an unconventional myosin implicated in cell adhesion and filopodial elongation. Indeed, pilot studies indicate that these proteins do contribute importantly to netrin-1-triggered intracellular signaling and regulating their functions, as inhibition of PITPalpha or Myo X attenuates netrin-1-induced neurite outgrowth and/or growth cone turning. Myo X also appears to be involved in delivering DCC to the cell periphery or neurites. Thus, the proposed studies are based on the hypothesis that PITPalpha and Myo X are involved in initiating DCC or neogenin signaling and functions. Specifically, we will: Aim 1) analyze the role of PITPalpha in netrin-1-induced phosphatidylinositol signaling and neurite outgrowth; Aim 2) investigate the role of PITPalpha in RGMa-induced neuronal survival; and, Aim 3) determine the role of Myo X in regulating DCC distribution and functions. We will take advantage of neurons expressing miRNA of PITPalpha or Myo X, neuron-specific Myo X conditional knock-out mice, and/or neurons with PITPalpha/Myo X expression knocked-down by in utero/ovo electroporation of their miRNAs in mouse/chicken embryos to assess these questions. Results of the proposed studies will provide insights into intracellular mechanisms controlling brain wiring. Defects in this process underlie neurologic disorders at all ages, from neonates to neurodegerative processes in the elderly. Understanding such signaling pathways provides a possible foundation for future novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: The long-term goal of this proposal is to understand molecular mechanisms underlying axon pathfinding, a process essential for brain wiring and functions. Defects in this process are implicated in early embryonic death, mental retardation, and impaired recovery of spinal cord injury.